Retrovirus mRNAs are normally polyadenylated within the proviral 3' long terminal repeat (LTR). The site of retrovirus transcript polyadenylation is flanked 3' by an LTR-specific sequence termed the U5 region, but the role of U5 in the determination of polyadenylation efficiency has not been addressed. We have used site-directed mutagenesis of a human immunodeficiency virus LTR to map U5 sequences which are required for efficient polyadenylation within the LTR. These LTR U5 region sequences display homology to a motif termed the G-T cluster, which is known to facilitate the efficient polyadenylation of mRNAs encoded by several cellular and viral genes. These results suggest that the LTR U5 region functions in vivo to permit efficient polyadenylation within the proviral 3' LTR.